Most lives keeps their genetic information in a form of DNA. The DNA is transcribed to messenger RNA (mRNA), and the mRNA is further translated to protein. In eukaryotic cells, some deletion of genetic information generally occur, when the DNA is processed to synthesize a matured mRNA. The deletion is caused by RNA splicing of intoron/exon, or protein processing. It is therefore understood that mRNA may be used for studying basic genetic analysis of protein structure, more advantageously than genomic DNA.
The mRNA is, however, very unstable, and may readily be decomposed by various ribonucleases. For this reason, DNA chain (cDNA) synthesized based on mRNA has been used as a material for the study. The cDNA is synthesized by using a target mRNA as a template, with the aid of a reverse transcriptase capable of synthesizing single-strand DNA.
Besides use of a promoter of a vector involved in transcription of mRNA (or, sense mRNA) containing a sequence capable of coding a polypeptide, it is also possible to synthesize an antisense mRNA transcribed from a complementary cDNA chain. The antisense mRNA is a powerful tool for understanding biological functions of proteins, and mRNA whose actions are remained unknown. The antisense RNA molecule can block production of a specific protein by inhibiting transcription, when annealed with a target mRNA. This type of inhibition of transcription is, therefore, supposed to be important for studying various gene products involved in various symptoms.
The single-strand cDNA is useful not only as a starting materials for synthesizing a double-strand cDNA with the aid of catalytic action of a DNA polymerase, but also as a template for polymerase chain reaction (referred to as PCR, hereinafter) According to this method, a specific genetic sequence may rapidly be amplified, using a plurality of nucleotide primers having reverse directionality, and a heat-resistant DNA polymerase. Copies of the target gene may rapidly be produced by repeating a cycle of annealing and DNA synthesis. In this way, the sense single-strand cDNA may be used for specifically amplifying segments of corresponding double-strand cDNA.
In the liquid phase process, thus-synthesized double-strand cDNA contains no marker which determines the directionality of the molecule. Therefore, newly synthesized double-strand cDNA clones are supposed to contain a 50% error in the direction of transcription, and cannot directly be inserted into a cloning vector. For this reason, there has been a demand for a technique of allowing insertion into vector under the correct directionality, and allowing rapid cloning of mRNA.
The single-strand cDNA or the double-strand cDNA may be synthesized also by using a solid phase (Non-Patent Document 1). In the general solid phase process, poly-deoxythymidylic acid (poly-dT) complementarily binds with the tail of polyadenylic acid (polyA) of a mRNA immobilized on a porous bead. Next, an antisense single-strand cDNA is synthesized based on the bound mRNA, with the aid of a reverse transcriptase. After digestive decomposition of the template RNA, a second cDNA chain is synthesized by a DNA polymerase. The synthesized double-strand cDNA has an antisense chain immobilized on the bead.
The solid phase process, however, cannot release the double-strand-cDNA product from the insoluble carrier. To avoid this problem, the double-strand cDNA product is heated so as to release the single-strand cDNA from the double-strand cDNA immobilized on the carrier, and the double-strand cDNA is synthesized by PCR using the single-strand cDNA. This, however, means a need of one additional step for the reaction, and a need of one appropriate set of primers for every PCR reaction.
Therefore, there has been a demand for a simple method of synthesizing a non-binding-type double-strand cDNA from an isolated mRNA.
On the other hand, there are known various methods of synthesizing the mRNA from the cDNA clone, one of which being the liquid phase process (Non-Patent Document 2). In this process, a double-strand cDNA is inserted to a vector having an RNA promoter. The vector is then digested by a restriction enzyme to produce a straight chain, and the mRNA is synthesized by an action of an RNA polymerase. The synthesized mRNA is treated with a DNase for removing the template DNA. If necessary, a polyadenylic acid tail is added in this process to the terminal of the newly-synthesized RNA, using a terminal transferase and dATP.
Also the solid phase synthesis process of mRNA is well known. One of them is described in Non-Patent Document 3. In this process, a DNA sequence is digested from a genome of bacteriophage lambda, to thereby produce a random DNA fragment having a sticky end. The sticky end is then blunted with a biotin-modified dUTP with the aid of T4 DNA polymerase.
In the process of DNA synthesis using T4 DNA polymerase, the restriction enzyme is selected so as to leave a sticky end having an exposed dA nucleotide so as to allow the biotin-modified dUTP to hybridize therewith. The random sequence is then immobilized onto an acrylamide carrier bound with avidin. The mRNA is synthesized from a sequence having a naturally-occurred lambda promoter sequence, using T7 RNA polymerase or SP6 RNA polymerase. This system is designed for the purpose of study based on kinetic analysis of transcription by bacteriophage lambda.
Both processes of liquid-phase and solid-phase syntheses of mRNA have drawbacks. The liquid-phase synthetic process is in need of using the RNA-promoter-containing vector, and the vector after being inserted must be converted into a straight-chain sequence. The solid-phase synthetic process does not always necessarily provide a complete genetic information. This is because the information is that of the immobilized genomic DNA, but not of mRNA. For this reason, there is a demand for an improved method of synthesizing mRNA.
Non-Patent Document 4 describes that an oligonucleotide covalently bound through an amino bond to a solid-phase carrier may conveniently be used for PCR experiment. The technique described in this document, however, does not specifically provide production of cDNA and RNA. Moreover, there is no description on a mechanism of removing the oligonucleotide covalently bound to the carrier.
Non-Patent Document 1 discloses a method by which an oligonucleotide is immobilized on a solid carrier, and cDNA is then synthesized on the solid surface using RNA as a template. Non-Patent Document 2 discloses a method by which RNA is immobilized on a carrier, and cDNA is synthesized using a reverse transcriptase.
The enzymatic elongation function of nucleic acid chain is, however, less likely to proceed between the solid surface and the liquid phase, leaving some problem in that whether the synthesized cDNA exactly reflects a state of expression of mRNA used as a template.    [Non-Patent Document 1] I. Raineri et al., Nucleic Acids Research, 19:4010, 1991    [Non-Patent Document 2] S. Shichijo et al., J. Neurosci. Res., 30:316-320, 1991    [Non-Patent Document 3] Hironori Terada, “Kotei-ka DNA niyoru Tensha no Doteki Kaiseki (Dynamic Analysis of Transcription by Immobilized DNA)”, Biophysics, 31:49-52, 1991    [Non-Patent Document 4] Stamm et al., Nucleic Acids Res., 19:1350, 1991    [Patent Document 1] Published Japanese Translation of PCT International Publication for Patent Application No. 8-500722    [Patent Document 2] Published Japanese Translation of PCT International Publication for Patent Application No. 2003-519482